B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting

Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. Here, we describe how DOCK8-deficient B cells are blocked at a light-zone checkpoint in the germinal centers of immunized mice, where they are unable to respond to T cell–dependent survival and selection signals, and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin upregulation, B–T cell conjugate formation, and costimulation are insufficient for sustained B and T cell activation when antigen availability is limited. Our findings provide an explanation for the failure of the humoral response in DOCK8 immunodeficiency syndrome and insight into how the level of available antigen modulates B–T cell cross-talk to fine-tune humoral immune responses and immunological memory.


Fig. S2 .
Fig. S2.Deep immunophenotyping of GC B cells using scRNASeq data (related to Fig. 2).(A) Stacked violin plots of differential expression between the Dock8 wt/wt and Dock8 cpm/cpm groups of the landmark genes across the 19 clusters at 0.9 resolution (see Fig. 2B).(B to E) GSEA between LZ2 and the other four LZ clusters to confirm their relative LZ, DZ, plasma cell or memory B cell signatures using previously published datasets, as indicated: (B) LZ2 versus LZ1 (the two clusters with highest Cd83 expression); (C) LZ2 versus LZ3; (D) LZ2 versus LZ4; and (E) LZ2 versus LZtoPrePC/Memory.

Fig. S3 .
Fig. S3.DOCK8-deficient GC B cells are blocked in the LZ and do not receive T cell help or differentiation signals.(A) Quantification of DZ:LZ ratio of CD45.2 + BCL2.Tg Dock8 wt/wt and Dock8 cpm/cpm splenic GC B cells as calculated from the scRNASeq dataset (see: Fig 2, D and E, and fig.S1, D and E).(B) Representative flow cytometry plots for DZ and LZ status (left), and quantification of the DZ:LZ ratios of CD45.2 + BCL2.Tg Dock8 wt/wt and Dock8 cpm/cpm splenic GC B cells (right).Data are representative of three experiments with 4-6 mice per group.(C and D) Dock8 wt/wt or Dock8 cpm/cpm CD45.2 + BCL2.Tg GFP + MD4 spleen B cells were adoptively transferred into CD45.1 recipients before immunization with SRBC-HEL.Representative images for the distribution of GFP + MD4 B cells (cyan), FDC-M2 expression in the LZ (yellow) and IgD expression on non-GC B cells (magenta) in the spleens of mice

Fig
Fig. S4.Effect of DOCK8 on B cell expression of phenotypic, activation and survival markers.Dock8 wt/wt or Dock8 cpm/cpm BCL2.Tg GFP + MD4 spleen B cells were adoptively transferred into Dock8 wt/wt C57BL/6 recipients before immunization with SRBC-HEL.(A) Surface expression of GC markers, (B) activation markers, (C) integrins and adhesion markers on CD95 + GL7 + IgM a+ GFP + HEL + splenic GC B cells after 8 days.(A to C) Data are pooled from two to three experiments with n = 4-9 mice per group per experiment.Bars indicate means with 95% confidence intervals and symbols represent individual mice.Unpaired twotailed t tests with Welch's correction were used for analysis with **P<0.01,***P<0.001,and ****P<0.0001.(D) Levels of the mouse BCL2 and the transgenic human BCL2 protein in splenic B cells from Dock8 wt/wt or Dock8 cpm/cpm non-transgenic and BCL2.Tg mice.Data are pooled from two experiments with n = 3 mice per group per experiment.Bars show means, error bars indicate 95% confidence intervals and circles indicate data from individual mice.Ordinary one-way ANOVA with Tukey's multiple-comparison test was used for statistical analyses.

Fig. S5 .
Fig. S5.DOCK8 is required for B and T cell proliferation when antigen is limiting.(A) CTV-labeled CD45.2 + Dock8 wt/wt or Dock8 cpm/cpm MD4 spleen B cells were loaded with indicated amounts of OVAHEL, mixed with CTV-labeled CD45.2 + OTII CD4 T cells and adoptively transferred into CD45.1 + recipients.Spleens were analyzed at 72 hours to assess proliferation of transferred CD45.2 + OTII CD4 T cells (left) and CD45.2 + MD4 B cells (right).Data are pooled from two experiments with n = 3-6 mice per group.(B) Illustration of the ex vivo model used to dissect B and T cell interactions.(C) Dose-dependent upregulation of activation markers on OTII CD4 T cells after 16 hours of culture with Dock8 wt/wt or Dock8 cpm/cpm splenic MD4 B cells and OVAHEL.(D) Proliferation of OTII CD4 T cells cultured with Dock8 wt/wt or Dock8 cpm/cpm MD4 B cells and 5 ng/ml of OVAHEL for indicated time points.(E) CD69 expression on OVAHEL-activated Dock8 wt/wt or Dock8 cpm/cpm MD4 B cells after 16 hours with OTII CD4 T cells.(F) Proliferation of Dock8 wt/wt or Dock8 cpm/cpm MD4 B cells at indicated time points after culture with OTII CD4 T cells and 5 ng/ml of OVAHEL.Data are pooled from N = 3-6 independent experiments with cells pooled from n = 2-6 mice per group.Symbols represent data from individual samples or mice, and bars show means with 95% confidence intervals.Data were analyzed by unpaired two-tailed t tests with Welch's correction and the Holm-Šídák method for multiple-comparisons, with *P<0.05,**P<0.01,***P<0.001,and ****P<0.0001.

Fig. S6 .
Fig. S6.Defective B-T cell costimulatory interactions at low antigen levels in the absence of DOCK8.(A to D) Dock8 wt/wt or Dock8 cpm/cpm splenic MD4 B cells and OTII CD4 T cells were cultured with indicated amounts of OVAHEL and anti-CD40 or anti-CD28 antibody for 96 hours.(A) T cell and (B) B cell proliferation at 96 hours with 5 ng/ml of OVAHEL and indicated amounts of anti-CD40 antibody (left) or heatmaps for (A) T cell and (B) B cell proliferation with indicated combinations of OVAHEL and anti-CD40 antibody (right).(C) T cell and (D) B cell proliferation at 96 hours with 5 ng/ml of OVAHEL and indicated amounts of anti-CD28 antibody (left) or heatmaps with indicated combinations of OVAHEL and anti-CD28 antibody (right).Circles indicate individual samples, bars show means with 95% confidence intervals.Cells were pooled from two mice per group in each experiment.Data are pooled from N = 2-3 experiments (bar graphs) or 1-3 experiments (heatmaps).Unpaired t tests with Welch's correction and the Holm-Šídák method for multiple-comparisons were used for analyses with *P<0.05,**P<0.01,and ***P<0.001.

Fig. S7 .
Fig. S7.B cells require DOCK8 to elicit T cell help but not for presentation of limiting amounts of soluble antigen.(A) Representative flow cytometry plots and pooled data from four experiments with two mice each for proliferation of OTII CD4 T cells cultured with Dock8 wt/wt or Dock8 cpm/cpm MD4 B cells and 500 ng/ml of indicated antibodies for 96 hours.Data analyzed using two-way ANOVA and Tukey's multiple-comparisons test with ****P<0.0001.(B) Representative flow cytometry plots and pooled data for Eα(52-68) peptide bound to I-A b on MD4 B cells after culture with HEL-EαGFP for 4 hours.Data are pooled from N = 3-7 independent experiments.(A and B) Circles denote data from individual mice and bars indicate means with 95% confidence intervals.(C and D) HEL(46-61) peptide bound

Fig. S8 .
Fig. S8.DOCK8 modulates B-T cell conjugate formation and actin cytoskeleton when antigen is limiting.(A) Representative images for B-T cell conjugate formation in the presence of 10 ng/ml of OVAHEL at indicated time points.Dock8 wt/wt MD4 B cells in blue with white boxes indicating conjugates, Dock8 cpm/cpm MD4 B cells in magenta with yellow boxes indicating conjugates and OTII CD4 T cells in green.(B) Distribution of T cell conjugates with Dock8 wt/wt or Dock8 cpm/cpm MD4 B cells at indicated B:T ratios.Data are representative of three experiments with cells pooled from n = 2-3 mice per group.Symbols indicate the proportion of B cells in each field of view that form conjugates. Bars show means with 95% confidence intervals.Data were analyzed by unpaired Welch's t test and the Holm-

Fig. S9 .
Fig. S9.Competition for T cell help within the GC is dependent on local antigen dose and modulated by DOCK8.Under physiological conditions, most antigens are of low affinity and abundance.Lower localized antigen levels within the GC elicit corresponding lower activation of B cells, reinforcing dependence on T cell help and ancillary pathways, such as those modulated by DOCK8.Maintaining antigen thresholds within the GC creates the competitive GC environment that supports selection and survival of the fittest B cell clones.